The goal of this application is to convene a three-day Workshop on the cutting-edge topic of Image Analysis in Three-Dimensional Cryo-Electron Microscopy slated for early spring 2015. This would follow the 1st workshop, which will be held from March 12-15, 2014 at the Granlibakken Conference Center at Lake Tahoe, CA. The goal of the workshop is to bring together specialists in algorithm development to assess the state of the art in image analysis approaches that tackle challenging biological problems and to discuss current limitations and road blocks in the 3D cryo-EM field as currently practiced. The workshop will provide an open forum for developers in this area, both established as well as students/postdocs to consider the detailed algorithms and methodologies in this fast developing field and identify areas in the most need of concerted effort. The central premise of the workshop is that gaining a comprehensive and quantitative understanding of highly sophisticated biological machines, complexes, and organelles of the cell requires the active, timely and coordinated development of complementary image analysis and data mining algorithms and approaches. The program consists of one keynote lecture, five platform sessions with both invited talks (70%), and talks selected from submitted abstracts (30%), as well as two poster sessions. All sessions will focus on interactive discussions with plenty of time for questions. The 2014 meeting will include a special Vision talk on the Past and future of image analysis methods by David DeRosier, Brandeis University. The talks will survey new method development efforts combined with details on advances within more traditional imaging approaches. The current platform sessions for the 2014 meeting are: Map Validation; Single Particle Approaches to Helical Symmetry and 2-D Crystals; Direct Detector Movie Processing; Tomogram Segmentation, Annotation and Filtration; Factors Limiting Resolution in Single Particle Analysis/Tomography; Heterogeneous Data in 2-D and 3-D. Drs. D. Hanein and S. Ludtke serve as Chairs of the meeting and Drs. W. Chiu (US), E. Egelman (US), B. Hankamer (Australia), M. Kikkawa (Japan), D. Mastronarde (US), P. Penczek (US), H. Stahlberg (Europe), F. Sun (China), and N. Volkmann (US) are members of the organizing committee. The Workshop will be extensively advertised and will be as inclusive and diversity-sensitive as the organizers can achieve and the 100-participant limit will allow.